scholarly journals The Real-Time-Based Assessment of the Microbial Killing by the Antimicrobial Compounds of Neutrophils

2011 ◽  
Vol 11 ◽  
pp. 2382-2390 ◽  
Author(s):  
J. T. Atosuo ◽  
E.-M. Lilius
Keyword(s):  
Real Time ◽  
Antimicrobial Agents ◽  
Hypochlorous Acid ◽  
Bacterial Cells ◽  
Photorhabdus Luminescens ◽  
Bacterial Luciferase ◽  
E Coli ◽  
High Concentrations ◽  
Time Basis ◽  
K 12

A recombinantEscherichia coliK-12 strain, transformed with a modified bacterial luciferase gene (luxABCDE) fromPhotorhabdus luminescens, was constructed in order to monitor the activity of various antimicrobial agents on a real-time basis. ThisE. coli-lux emitted, without any addition of substrate, constitutive bioluminescence (BL), which correlated to the number of viable bacterial cells. The decrease in BL signal correlated to the number of killed bacterial cells. Antimicrobial activity of hydrogen peroxide (H2O2) and myeloperoxidase (MPO) was assessed. In high concentrations, H2O2alone had a bacteriocidic function and MPO enhanced this killing by forming hypochlorous acid (HOCl). Taurine, the known HOCl scavenger, blocked the killing by MPO. WhenE. coli-lux was incubated with neutrophils, similar killing kinetics was recorded as in H2O2/MPO experiments. The opsonization of bacteria enhanced the killing, and the maximum rate of the MPO release from lysosomes coincided with the onset of the killing.

Transcription Analysis of stx1, marA, and eaeA Genes in Escherichia coli O157:H7 Treated with Sodium Benzoate

2008 ◽  
Vol 71 (7) ◽  
pp. 1469-1474 ◽  
Author(s):  
FAITH J. CRITZER ◽  
DORIS H. D'SOUZA ◽  
DAVID A. GOLDEN
Keyword(s):  
Escherichia Coli ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Sodium Benzoate ◽  
Antimicrobial Agents ◽  
Transcription Analysis ◽  
E Coli ◽  
Reverse Transcriptase Pcr ◽  
One Step ◽  
K 12

Expression of the multiple antibiotic resistance (mar) operon causes increased antimicrobial resistance in bacterial pathogens. The activator of this operon, MarA, can alter expression of >60 genes in Escherichia coli K-12. However, data on the expression of virulence and resistance genes when foodborne pathogens are exposed to antimicrobial agents are lacking. This study was conducted to determine transcription of marA (mar activator), stx1 (Shiga toxin 1), and eaeA (intimin) genes of E. coli O157:H7 EDL933 as affected by sodium benzoate. E. coli O157:H7 was grown in Luria-Bertani broth containing 0 (control) and 1% sodium benzoate at 37°C for 24 h, and total RNA was extracted. Primers were designed for hemX (209 bp; housekeeping gene), marA (261 bp), and eaeA (223 bp) genes; previously reported primers were used for stx1. Tenfold dilutions of RNA were used in a real-time one-step reverse transcriptase PCR to determine transcription levels. All experiments were conducted in triplicate, and product detection was validated by gel electrophoresis. For marA and stx1, real-time one-step reverse transcriptase PCR products were detected at a 1-log-greater dilution in sodium benzoate–treated cells than in control cells, although cell numbers for each were similar (7.28 and 7.57 log CFU/ml, respectively). This indicates a greater (albeit slight) level of their transcription in treated cells than in control cells. No difference in expression of eaeA was observed. HemX is a putative uroporphyrinogen III methylase. The hemX gene was expressed at the same level in control and treated cells, validating hemX as an appropriate housekeeping marker. These data indicate that stx1 and marA genes could play a role in pathogen virulence and survival when treated with sodium benzoate, whereas eaeA expression is not altered. Understanding adaptations of E. coli O157:H7 during antimicrobial exposure is essential to better understand and implement methods to inhibit or control survival of this pathogen in foods.

Real-time activity assays of β-lactamases in living bacterial cells: application to the inhibition of antibiotic-resistant E. coli strains

2017 ◽  
Vol 13 (11) ◽  
pp. 2323-2327 ◽  
Author(s):  
Ying Ge ◽  
Ya-Jun Zhou ◽  
Ke-Wu Yang ◽  
Yi-Lin Zhang ◽  
Yang Xiang ◽  
...  
Keyword(s):  
Real Time ◽  
Biological Systems ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Time Activity ◽  
E Coli ◽  
Living Bacteria

A UV-Vis approach is reported for activity assays and inhibition of β-lactamases in complex biological systems of living bacteria.

scholarly journals Waterborne illness and injury: a feasibility study of a site-specific predictive model for beach water quality at Beachway Park in the City of Burlington

2021 ◽  
Author(s):  
Ramien Sereshk
Keyword(s):  
Water Quality ◽  
Predictive Model ◽  
Real Time ◽  
Site Specific ◽  
E Coli ◽  
Time Basis ◽  
A Site ◽  
Persistence Model ◽  
Beach Water Quality ◽  
Beach Water

It is commonly assumed that the persistence model, using day-old monitoring results, will provide accurate estimates of real-time bacteriological concentrations in beach water. However, the persistence model frequently provides incorrect results. This study: 1. develops a site-specific predictive model, based on factors significantly influencing water quality at Beachway Park; 2. determines the feasibility of the site-specific predictive model for use in accurately predicting near real-time E. coli levels. A site-specific predictive model, developed for Beachway Park, was evaluated and the results were compared to the persistence model. This critical performance evaluation helped to identify the inherent inaccuracy of the persistence model for Beachway Park, which renders it an unacceptable approach for safeguarding public health from recreational water-borne illnesses. The persistence model, supplemented with a site-specific predictive model, is recommended as a feasible method to accurately predict bacterial levels in water on a near real-time basis.

scholarly journals HypVW is an HOCl-Sensing Two Component System in Escherichia coli

2021 ◽  
Author(s):  
Sara El Hajj ◽  
Camille Henry ◽  
Alexandra Vergnes ◽  
Laurent Loiseau ◽  
Brasseur Gael ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Hypochlorous Acid ◽  
Methionine Sulfoxide ◽  
Methionine Sulfoxide Reductase ◽  
Bacterial Cells ◽  
E Coli ◽  
Two Component ◽  
Redox Switch ◽  
Two Component Systems ◽  
Methionine Residues

Two component systems (TCS) are signalling pathways that allow bacterial cells to sense, respond and adapt to fluctuating environments. Among the classical TCS of Escherichia coli, YedVW has been recently showed to be involved in the regulation of msrPQ, encoding for the periplasmic methionine sulfoxide reductase system. In this study, we demonstrate that hypochlorous acid (HOCl) induces the expression of msrPQ in a YedVW dependant manner, whereas H2O2, NO and paraquat (a superoxide generator) do not. Therefore, YedV appears to be an HOCl-sensing histidine kinase. Based on this finding, we proposed to rename this system HypVW.  Moreover, using a directed mutagenesis approach, we show that Met residues located in the periplasmic loop of HypV (formerly YedV) are important for its activity. Given that HOCl oxidizes preferentially Met residues, we bring evidences that HypV could be activated via the reversible oxidation of its methionine residues, thus conferring to MsrPQ a role in switching HypVW off. Based on these results, we propose that the activation of HypV by HOCl could occur through a Met redox switch. HypVW appears to be the first characterized TCS able to detect HOCl in E. coli. This study represents an important step in understanding the mechanisms of reactive chlorine species resistance in prokaryotes.

scholarly journals Strain improvement of Escherichia coli K-12 for recombinant production of deuterated proteins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vinardas Kelpšas ◽  
Claes von Wachenfeldt
Keyword(s):  
Escherichia Coli ◽  
Structural Biology ◽  
Strain Improvement ◽  
Growth Media ◽  
Genomic Changes ◽  
E Coli ◽  
Recombinant Production ◽  
High Concentrations ◽  
Neutron Protein Crystallography ◽  
K 12

AbstractDeuterium isotope labelling is important for structural biology methods such as neutron protein crystallography, nuclear magnetic resonance and small angle neutron scattering studies of proteins. Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affect growth of cells. The generation time for Escherichia coli K-12 in deuterated medium is substantially increased compared to cells grown in hydrogenated (protiated) medium. By using a mutagenesis plasmid based approach we have isolated an E. coli strain derived from E. coli K-12 substrain MG1655 that show increased fitness in deuterium based growth media, without general adaptation to media components. By whole-genome sequencing we identified the genomic changes in the obtained strain and show that it can be used for recombinant production of perdeuterated proteins in amounts typically needed for structural biology studies.

scholarly journals Phage Mediate Bacterial Self Recognition

2018 ◽  
Author(s):  
Sooyeon Song ◽  
Yunxue Guo ◽  
Jun-Seob Kim ◽  
Xiaoxue Wang ◽  
Thomas K. Wood
Keyword(s):  
Kin Recognition ◽  
Group Behavior ◽  
Lytic Phage ◽  
Bacterial Cells ◽  
Demarcation Line ◽  
E Coli ◽  
Self Recognition ◽  
The Family ◽  
K 12

AbstractCells are social, and self-recognition is an important and conserved aspect of group behavior where cells assist kin and antagonize non-kin to conduct group behavior such as foraging for food and biofilm formation. However, the role of the common bacterial cohabitant, phage, in kin recognition, has not been explored. Here we find that a boundary (demarcation line) is formed between different swimmingEscherichia colistrains but not between identical clones; hence, motile bacterial cells discriminate between self and non-self. The basis for this self-recognition is a novel, 49 kb, T1-type, lytic phage of the family siphoviridae (named here SW1) that controls formation of the demarcation line by utilizing one of the host’s cryptic prophage proteins, YfdM, to propagate. Critically, SW1 increases the fitness ofE. coliK-12 compared to the identical strain that lacks the phage. Therefore, bacteria use phage to recognize kin.

scholarly journals Metabolic Flux Analysis of Catechin Biosynthesis Pathways Using Nanosensor

Antioxidants ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 288
Author(s):  
Habiba Kausar ◽  
Ghazala Ambrin ◽  
Mohammad K. Okla ◽  
Walid Soufan ◽  
Abdullah A. Al-Ghamdi ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Real Time ◽  
Metabolic Flux Analysis ◽  
Metabolic Flux ◽  
Regulatory Element ◽  
Flux Analysis ◽  
Bacterial Cells ◽  
Biosynthesis Pathway ◽  
Real Time Monitoring ◽  
E Coli

(+)-Catechin is an important antioxidant of green tea (Camelia sinensis (L.) O. Kuntze). Catechin is known for its positive role in anticancerous activity, extracellular matrix degradation, cell death regulation, diabetes, and other related disorders. As a result of enormous interest in and great demand for catechin, its biosynthesis using metabolic engineering has become the subject of concentrated research with the aim of enhancing (+)-catechin production. Metabolic flux is an essential concept in the practice of metabolic engineering as it helps in the identification of the regulatory element of a biosynthetic pathway. In the present study, an attempt was made to analyze the metabolic flux of the (+)-catechin biosynthesis pathway in order to decipher the regulatory element of this pathway. Firstly, a genetically encoded fluorescence resonance energy transfer (FRET)-based nanosensor (FLIP-Cat, fluorescence indicator protein for (+)-catechin) was developed for real-time monitoring of (+)-catechin flux. In vitro characterization of the purified protein of the nanosensor showed that the nanosensor was pH stable and (+)-catechin specific. Its calculated Kd was 139 µM. The nanosensor also performed real-time monitoring of (+)-catechin in bacterial cells. In the second step of this study, an entire (+)-catechin biosynthesis pathway was constructed and expressed in E. coli in two sets of plasmid constructs: pET26b-PT7-rbs-PAL-PT7-rbs-4CL-PT7-rbs-CHS-PT7-rbs-CHI and pET26b-T7-rbs-F3H-PT7-rbs- DFR-PT7-rbs-LCR. The E. coli harboring the FLIP-Cat was transformed with these plasmid constructs. The metabolic flux analysis of (+)-catechin was carried out using the FLIP-Cat. The FLIP-Cat successfully monitored the flux of catechin after adding tyrosine, 4-coumaric acid, 4-coumaroyl CoA, naringenin chalcone, naringenin, dihydroquercetin, and leucocyanidin, individually, with the bacterial cells expressing the nanosensor as well as the genes of the (+)-catechin biosynthesis pathway. Dihydroflavonol reductase (DFR) was identified as the main regulatory element of the (+)-catechin biosynthesis pathway. Information about this regulatory element of the (+)-catechin biosynthesis pathway can be used for manipulating the (+)-catechin biosynthesis pathway using a metabolic engineering approach to enhance production of (+)-catechin.

scholarly journals Transferable high-level trimethoprim resistance among isolates ofEscherichia colifrom urinary tract infections in Ontario, Canada

1992 ◽  
Vol 109 (3) ◽  
pp. 473-481 ◽  
Author(s):  
N. Harnett
Keyword(s):  
Nalidixic Acid ◽  
Antimicrobial Agents ◽  
Acid Resistance ◽  
The Public ◽  
E Coli ◽  
High Level ◽  
Tract Infections ◽  
K 12 ◽  
Level Resistance ◽  
Nalidixic Acid Resistance

SUMMARYOf 1171 isolates ofEscherichia coliisolated from urine samples at the Public Health Laboratory, Toronto, Ontario, Canada, between May 1990 and December 1991, 120 (10·3%) were resistant to trimethoprim (TMP), cotrimoxazole (TMP/SMX), sulfamethoxazole (SMX) and other antimicrobial agents; 110 of the 120 isolates (91·7%) were resistant to four or more agents. The majority of resistant isolates (91·7%) exhibited high-level resistance (MIC > 1000 mg/L) to TMP. The MIC of TMP/SMX for all 120 isolates was > 2·0/38·0 mg/L and for SMX > 1024 mg/L. High-level resistances were also present among the β-lactam antimicrobials with MICs ranging from 16- > 256 mg/L. Forty-three of 120 TMP-resistant (35·8%) isolates conjugally transferred TMP-resistance toE. coliK-12. Co-transfer of several other resistances was observed. SMX cotransferred from 86% of the 43 donors and β-lactams together with SMX cotransferred from 70%. Nalidixic acid resistance was present among 22 (18·3%) of the 120 resistant isolates, however, nalidixic acid resistance was not transferred toE. coliK-12.

Real-time PCR and enzyme-linked fluorescent assay methods for detecting Shiga-toxin-producingEscherichia coliin mincemeat samples

2007 ◽  
Vol 53 (3) ◽  
pp. 337-342 ◽  
Author(s):  
A. Stefan ◽  
S. Scaramagli ◽  
R. Bergami ◽  
C. Mazzini ◽  
M. Barbanera ◽  
...  
Keyword(s):  
Real Time ◽  
Shiga Toxin ◽  
Real Time Pcr ◽  
Central Italy ◽  
Health Concern ◽  
Bacterial Cells ◽  
Fluorescent Assay ◽  
Point Of Sale ◽  
E Coli ◽  
Assay Methods

This work aimed to compare real-time polymerase chain reaction (PCR) with the commercially available enzyme-linked fluorescent assay (ELFA) VIDAS ECOLI O157™ for detecting Escherichia coli O157 in mincemeat. In addition, a PCR-based survey on Shiga-toxin-producing E. coli (STEC) in mincemeat collected in Italy is presented. Real-time PCR assays targeting the stx genes and a specific STEC O157 sequence (SILO157, a small inserted locus of STEC O157) were tested for their sensitivity on spiked mincemeat samples. After overnight enrichment, the presence of STEC cells could be clearly determined in the 25 g samples containing 10 bacterial cells, while the addition of five bacteria provided equivocal PCR results with Ct values very close to or above the threshold of 40. The PCR tests proved to be more sensitive than the ELFA-VIDAS ECOLI O157™, whose detection level started from 50 bacterial cells/25 g of mincemeat. The occurrence of STEC in 106 mincemeat (bovine, veal) samples collected from September to November 2004 at five different points of sale in Italy (one point of sale in Arezzo, Tuscany, central Italy, two in Mantova, Lombardy, Northern Italy, and two in Bologna, Emilia-Romagna, upper-central Italy) was less than 1%. Contamination by the main STEC O-serogroups representing a major public health concern, including O26, O91, O111, O145, and O157, was not detected. This survey indicates that STEC present in these samples are probably not associated with pathogenesis in humans.

scholarly journals Apple Flavonoid Phloretin Inhibits Escherichia coli O157:H7 Biofilm Formation and Ameliorates Colon Inflammation in Rats

2011 ◽  
Vol 79 (12) ◽  
pp. 4819-4827 ◽  
Author(s):  
Jin-Hyung Lee ◽  
Sushil Chandra Regmi ◽  
Jung-Ae Kim ◽  
Moo Hwan Cho ◽  
Hyungdon Yun ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Trinitrobenzene Sulfonic Acid ◽  
Content Type ◽  
E Coli ◽  
Colon Inflammation ◽  
K 12

ABSTRACTPathogenic biofilms have been associated with persistent infections due to their high resistance to antimicrobial agents, while commensal biofilms often fortify the host's immune system. Hence, controlling biofilm formation of both pathogenic bacteria and commensal bacteria is important in bacterium-related diseases. We investigated the effect of plant flavonoids on biofilm formation of enterohemorrhagicEscherichia coliO157:H7. The antioxidant phloretin, which is abundant in apples, markedly reducedE. coliO157:H7 biofilm formation without affecting the growth of planktonic cells, while phloretin did not harm commensalE. coliK-12 biofilms. Also, phloretin reducedE. coliO157:H7 attachment to human colon epithelial cells. Global transcriptome analyses revealed that phloretin repressed toxin genes (hlyEandstx2), autoinducer-2 importer genes (lsrACDBF), curli genes (csgAandcsgB), and dozens of prophage genes inE. coliO157:H7 biofilm cells. Electron microscopy confirmed that phloretin reduced fimbria production inE. coliO157:H7. Also, phloretin suppressed the tumor necrosis factor alpha-induced inflammatory responsein vitrousing human colonic epithelial cells. Moreover, in the rat model of colitis induced by trinitrobenzene sulfonic acid (TNBS), phloretin significantly ameliorated colon inflammation and body weight loss. Taken together, our results suggest that the antioxidant phloretin also acts as an inhibitor ofE. coliO157:H7 biofilm formation as well as an anti-inflammatory agent in inflammatory bowel diseases without harming beneficial commensalE. colibiofilms.

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